1. Field of the Invention
The present invention relates to a method for selecting or obtaining potyvirus-resistant plants. The method is particularly applicable to plants of the family of Solanaceae, Cucurbitaceae, Cruciferae and Compositae. The invention also comprises the sequences for conferring potyvirus resistance and/or for labeling the genes for resistance or for sensitivity to these potyviruses.
2. Description of the Related Art
The potyvirus group, the standard member of which is PVY, for potato virus Y, is the largest plant virus group. In fact, potyviruses are capable of infecting more than 30 plant families currently registered. This group comprises at least 180 members, which corresponds to a third of the plant viruses currently known. Potyvirus transmission is effected by aphids (for example Myzus persicae) via the nonpersistent mode. The symptoms caused by potyviruses are abnormalities in leaf coloration (mosaics, yellowing of veins), leaf deformities, vein necroses which can result in necrosis of the entire plant, and considerable decreases in size of the sick plant, greatly influencing productivity.
The Solanaceae, Cucurbitaceae, Cruciferae and Compositae are particularly sensitive to potyviruses. The Solanaceae, and more particularly the tomato and the capsicum (or pepper), are infected with at least seven different potyviruses throughout the world: potato virus Y (PVY) is present on all the growing areas, whereas the others are confined to a continent (tobacco etch virus, pepper mottle virus and Peru tomato virus on the American continent, pepper veinal mottle virus and potyvirus E in Africa, and chili veinal mottle virus in Asia). This compartmentalization is, however, no longer absolute, several potyviruses having been identified outside their area of origin. In France, and more particularly in the Mediterranean basin, the predominant potyvirus is PVY. Having appeared in the 1970s, PVY epidemics developed in crops in the open field and then in crops under cover, where, from 1982, new PVY isolates were demonstrated, which caused particularly serious symptoms of necrosis in the tomato (Gëbre-Selassie et al., 1987). For some of these potyviruses, it is possible to classify the isolates according to their ability to circumvent resistance alleles. This is the case of PVY with respect to the pvr2 gene in the capsicum, the only resistance gene that has been used for a long time by selectors, but that is circumvented in the Mediterranean region and in tropical regions. Despite the predominance of PVY in France, the internationalization of the seed market makes it necessary for selectors who sell their seeds abroad to use genes controlling resistance to these various potyviruses. More generally, considering the economical importance of potyvirus infections and the lack of direct means for combating this type of infection, the search for resistant plant varieties constitutes one of the main lines of plant improvement.
Potyviruses have a nonenveloped filamentous structure (Langenberg and Zhang, 1997), from 680 to 900 nm long and from 11 to 15 nm wide (Dougherty and Carrington, 1988; Riechmann et al., 1992). The viral genome consists of a single-stranded sense RNA approximately 10 kb in length. The single-stranded RNA has a poly A tail at its 3′ end and binds, in the 5′ position to a viral protein called VPg (Murphy et al., 1990, Takahashi et al., 1997). The viral RNA encodes 10 proteins involved in polyproteins cleavage, genome replication, cell-to-cell movement and long-distance movement, transmission via aphids, etc. Virus control can only be carried out indirectly. Specifically, it is only possible to eliminate the vector of the disease (the aphids in this case) or to grow varieties resistant to the viral infection and/or to the vectors.
Faced with an attack from a pathogen (viruses, bacteria, fungi or nematodes), the plant has several strategies for defending itself or withstanding infection. Among the defense strategies, the plant can set up:                systems of mechanical defense by developing and reinforcing physical barriers consisting of a thick cuticle on the leaves and/or a deposit of callose or of lignins on the cell walls. Thus, the entry of pathogens into and their movement in the plant are made more difficult;        systems of chemical or biochemical defense by synthesizing toxic compounds such as, for example, tannins, phytoalexins and various protein complexes.        
Among the resistance strategies, non-host resistance (when all the entities of a species are resistant to a given pathogen) is distinguished from host resistance (when at least one entity of the species is sensitive to a strain of the pathogenic agent). Host resistance, the most well-known and the most well-characterized to date, is that which involves a major, dominant gene. When the major gene is in the presence of a gene specific for avirulence of the pathogenic agent, the incompatibility between the plant and the pathogen is set up and the plant is resistant. This interaction, described by Flor (1955), is also called “gene-to-gene” model and is very often associated with localized necrosis of the plant tissue at the site of infection (hypersensitivity reaction). Although quite widespread, this “gene-to-gene” model is not universal because some systems of resistance described do not function according to this model, the differences lying in particular in the mode of action of the resistance gene. Genes exist which are recessive, which are superdominant or which exert incomplete dominance. Several avirulence genes can interact with the same resistance gene. Many resistances are also polygenic, several genes present in the plant are then involved in the resistance, each of them having a partially protective effect and being able to control different mechanisms.
To date, many dominant genes according to the “gene-to-gene” model have been cloned. They have related gene structures, although they act against varied pathogenic agents (viruses, fungi, bacteria, insects, nematodes). The presence of conserved domains has made it possible to define 4 major classes (Hammond-Kosack and Jones, 1997) of dominant genes.
Singularly, it is estimated that 40% of potyvirus resistances are recessive, whereas, in the other viral groups, this proportion only reaches 20% on average. Fraser (1992) has put forward the hypothesis that recessive resistances are different from dominant resistances of “gene-to-gene” type and are the result of a specific deficiency or alteration in the product of a host's gene, necessary for accomplishing the viral cycle in the plant. The dominant alleles for sensitivity are therefore thought to correspond to the availability of this product involved in the plant/pathogen interactions.
It has been shown that point mutations in the viral gene encoding the VPg protein are involved in the circumventing of potyvirus resistance in several host-pathogen couples. This has been shown in the TVMV/Nicotiana tabacum (va gene), PVY/tomato (pot-1 gene), LMV/lettuce (mol gene) and PSbMV/pea (sbm1 gene) couples (Keller et al., 1998, Morel, 2001, Redondo, 2001 Nicholas et al., 1997). This does not exclude the fact that other viral genes may also be involved.
Moreover, Wittman et al. (1997) have shown that an isoform of eukaryotic translation initiation factor eIF4E from Arabidopsis thaliana interacts with the viral protein VPg of the turnip mosaic virus (TuMV). This same interaction has been detected between TEV VPg and eIF4E from tobacco and from tomato (Schaad et al., 2000).
The eIF4E gene encodes a eukaryotic RNA translation initiation factor. eIF4E corresponds to one of the subunits of the translation factor eIF4F (in wheatgerm, it corresponds to the p26 subunit). The eIF4E translation factor binds to the mRNA cap at the m7Gs. The structure of eIF4E is characterized by a region rich in tryptophan residues (10 in Arabidopsis thaliana, 11 in wheat and 12 in mammals). These tryptophan residues are thought to be involved in binding to the m7G functional group (Rudd, K. et al. 1998). The eIF4E translation factor is encoded by a multigene family. For example, in Arabidopsis thaliana, 4 copies of eIF4E have been identified (Rodriguez et al., 1998, Robaglia et al., personal communication). These copies exhibit, in pairs, between 44 and 82% identity.
All these studies make reference to the correlation between the eIF4E/VPg interaction and the sensitivity of the plant to potyviruses, but none emphasize, or even suggest, that this interaction could result in resistance. On the contrary, it is even indicated in Schaad et al., 2000, that the VPg/eIF4E interaction does not play a role in resistance since the genetic determinants of the VPa/eIF4E interaction are different from those which allow potyviruses (via VPg) to circumvent the resistance.
It is therefore to the inventors' credit, in such a state of the art, to have demonstrated eIF4E proteins, and also the corresponding genes, involved in plant resistance or sensitivity to potyviruses.
The inventors have in particular noted that various potyvirus-resistant plants have point mutations located in the same region of the eIF4E protein; this region, which is very conserved between the eIF4E proteins derived from various plant species, in particular from Solanaceae, is derined by the gene sequence (I) (SEQ ID NO: 1) below:    DX1X2X3X4KSX5QX6AWGSSX7RX8X9YTFSX10VEX11FWX12X13YNNIHX14PSKLX15X16GAD            in which:        X1, X2, X3, X4, X6, X7, X8, X9, X10, X12, X13, X15 and X16 each represent a neutral amino acid;        X5 and X14 represent a basic amino acid;        X11 represents an acidic amino acid;        D, K, S, Q, A, W, G, R, Y, T, F, V, E, N, I, H, P and L have their usual 1-letter code meaning.        
The term “neutral amino acid” is here defined as any amino acid chosen from the following: alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine, glycine, serine, threonine, tyrosine, cysteine, glutamine, asparagine. The term “charged amino acid” is defined as any amino acid chosen from the following: histidine, lysine, arginine, glutamate and aspartate. Among these charged amino acids, histidine, lysine and arginine are basic amino acids and glutamate and aspartate are acidic amino acids.
The sequence (I) is also represented in the sequence listing in the appendix under the number SEQ ID NO: 1.
The mutations demonstrated by the inventors in the pepper are as follows:                substitution of the neutral amino acid X3 of sequence (I) with a basic amino acid;        substitution of the neutral amino acid X7 of sequence (I) with a basic amino acid;        substitution of the aspartate residue in the C-terminal position of sequence (I) with a neutral amino acid.        
The mutation of X3 was observed in pepper lines exhibiting two different types of potyvirus resistance (pvr21 and pvr22); the peppers exhibiting the pvr21 phenotype also having the mutation at position X7, and the peppers exhibiting the pvr22 phenotype also having the mutation in the C-terminal position.
In the tomato, the inventors have in particular observed the following mutations:                substitution of the neutral amino acid X1 of sequence (I) with a basic amino acid;        substitution of the Ala residue of the AWGSS unit of sequence (I) with an acidic amino acid.        
Due to the very high degree of sequence conservation of the eIF4E genes in eukaryotes and to the availability of 3D structure for the mouse and yeast eIF4E proteins (Marcotrigiano et al., 1997, Cell 89: 951-961; Matsuo et al., 1997, Nat. Struct. Biol. 4: 717-724), the positions of the mutations with respect to the 3D structure of eIF4E in the capsicum and the tomato can be determined. All these mutations are physically close and at the surface of the protein. Moreover, these mutations do not involve amino acids that are very conserved in eukaryotes, nor those involved in the essential functions of eIF4E, namely cap recognition or interaction between eIF4E and eIF4G or the 4E-binding protein.
However, it is probable that these mutations exert an effect on the VPg/eIF4E interaction by modification of the structure of eIF4E in the region(s) thereof involved in this interaction. This structural modification probably results from the substitution of amino acids with amino acids having a different charge (replacement of neutral amino acids with charged amino acids, or, conversely, of charged amino acids with neutral amino acids, or amino acids having an opposite charge), which constitutes the point common to all the mutations demonstrated by the inventors. It can therefore reasonably be assumed that other mutations of the same type in a plant eIF4E protein, in the region defined by sequence (I), will lead to similar structural modifications, producing the same effect on the VPa/eIF4E interaction.
In particular, it appears that the substitution of at least one of the neutral amino acids, X1, X2, X3 or X4 with a charged amino acid, in particular with a basic amino acid, plays an important role in potyvirus resistance.
These observations makes it possible to propose tools, in particular genetic tools, for screening and/or for obtaining plants resistant or sensitive to potyviruses.
The present invention relates more particularly to a method for selecting potyvirus-resistant plants, characterized in that it comprises the detection, in the plants to be tested:                of the presence or of the absence of an eIF4E protein (hereinafter called: “wild-type eIF4E protein”) comprising a region defined by sequence (I) above, or of a sequence encoding said protein;        of the presence or of the absence of a mutant eIF4E protein comprising a region derived from that defined by sequence (I) above, by substitution of at least one neutral amino acid of said sequence (I) with a charged amino acid, preferably a basic amino acid, and/or substitution of at least one charged amino acid of said sequence (I) with a neutral amino acid or an amino acid having an opposite charge, or of a sequence encoding said protein;        and the selection of the plants in which a mutant eIF4E protein or a sequence encoding said protein is detected, and in which no wild-type eIF4E protein or sequence encoding said protein is detected.        
A subject of the present invention is also a method for selecting plants which can be used for obtaining potyvirus-resistant plants, characterized in that it comprises the detection, in the plants to be tested, of the presence or of the absence of the mutant eIF4E protein as defined above or of a sequence encoding said protein, and the selection of the plants in which said mutant eIF4E protein or a sequence encoding said protein is detected.
According to a preferred embodiment of the invention, said mutant eIF4E protein comprises a region derived from that defined by sequence (I) above, by:                a) substitution of at least one of the amino acids X1, X2, X3 or X4 of said sequence (I) with a charged amino acid, and        b) substitution of at least one of the other neutral amino acids of said sequence (I) with a charged amino acid and/or substitution of at least one charged amino acid of said sequence (I) with a neutral amino acid or an amino acid having an opposite charge.        
The detection of the presence or of the absence of a wild-type or mutant eIF4E protein can be carried out in particular using antibodies specifically directed against the desired form of the eIF4E protein. They may in particular be antibodies directed either against the wild-type form or against the mutant form of the region of eIF4E defined by sequence (I).
For the detection of the presence or of the absence of a sequence encoding a wild-type eIF4E protein or of a sequence encoding a mutant eIF4E protein, many tools are available; they may in particular be polynucleotides derived from the sequence of the eIF4E gene, and in particular polynucleotides capable of hybridizing selectively either with a wild-type allele or with a mutant allele of eIF4E, as defined above, or polynucleotides which make it possible to amplify the region of eIF4E containing the desired mutation; they may also be restriction enzymes which recognize a target sequence present in the wild-type form but not in the mutated form (or vice versa).
A subject of the present invention is thus the use of a selection tool chosen from:                a) a polynucleotide encoding a wild-type or mutant eIF4E protein as defined above;        b) a polynucleotide complementary to the polynucleotide a);        c) an fragment of at least 10 bp of a polynucleotide a) or b);        d) an antibody directed against a wild-type or mutant eIF4E protein as defined above;for selecting potyvirus-resistant plants.        
In particular, the invention relates to a method for selecting potyvirus-resistant plants, characterized in that it comprises the use of at least one means of selection chosen from the group of genetic tools (or similar) comprising:
*A/ all or part of one at least of the sequences selected from the subgroup comprising:
                SEQ ID NO: 2        SEQ ID NO: 4        SEQ ID NOs: 6 and 8,        any analog of these sequences resulting from the degeneracy of the genetic code,        any cDNA sequence complementary to at least one of these sequences and/or to at least one of their analogs;*B/ all or part of one at least of the products of transcription of the sequences A;*C/ all or part of one at least of the products of translation of the sequences A;*D/ all or part of at least one antibody specific for at least one product of translation of C/;*E/ and any combination of tools A, B, C and D.        
Preferably, the means of selection are selected from the tool subgroups A/ and/or B/, and even more preferably from the tool subgroup A/.
By means of this simple, easy and reliable pinpointing of plants resistant or sensitive to potyviruses, the inventors have thus developed a new method based on the use of sequences corresponding to the eIF4E gene.
The method which is the subject of the invention applies particularly to the Solanaceae, Cucurbitaceae, Cruciferae and Compositae, and more particularly to plants of the genera Lycopersicon, Capsicum, Nicotiana, Solanum, Lactuca, Cucumis, Arabidopsis, etc.
The potyviruses concerned are, for example, potato virus Y (PVY), tobacco etch virus (TEV) and/or lettuce mosaic virus (LMV) and/or zucchini yellow mosaic virus (ZYMV) and/or turnip mosaic virus (TuMV).
To implement the method according to the invention, use is made of nucleotide sequences and/or peptide sequences or restriction enzymes as means of detection, probes or primers, for selecting plants resistant or sensitive to potyviruses.
These means of detection comprise in particular nucleotide probes or primers.
For the purpose of the present invention, the term “primer” is intended to mean any polynucleotide sequence which can be used for amplifying a sequence of an eIF4E gene liable to comprise a mutation associated with potyvirus resistance. They are in particular polynucleotides which can be used for amplifying all or part of the sequence of eIF4E encoding the region of eIF4E defined by sequence (I), or of the mutant sequence which is derived therefrom.
For the purpose of the present invention, the term “probe” is intended to mean any polynucleotide sequence which hybridizes with a wild-type eIF4E gene or with a mutant eIF4E gene as defined above. This includes in particular the nucleotide sequences capable of hybridizing selectively either with an allele of the eIF4E gene associated with potyvirus resistance, or with an allele of the eIF4E gene associated with potyvirus sensitivity.
These probes and these primers can be used as markers specific for the plants resistant or sensitive to potyviruses.
In accordance with the invention, it is possible to sort the potyvirus-sensitive plants from the potyvirus-resistant plants by means of the genetic tools (or similar) (A) to (E), or even specific restriction enzymes. The latter will be described below.
The nucleotide sequences (A) SEQ ID NOs: 2, 4, 6 and 8 correspond to different Solanaceae eIF4E genes involved in potyvirus resistance encoding a eukaryotic RNA translation initiation factor. SEQ ID NO: 8 corresponds to a recessive eIF4E allele for resistance to a potyvirus, while SEQ ID NOs: 2, 4, and 6 represent dominant eIF4E alleles for sensitivity to a potyvirus.
Means of selection or genetic markers for resistance or for sensitivity to potyviruses have therefore been discovered in accordance with the invention. The method of selection according to the invention can involve, separately or together, the two types of means of selection or markers.
Naturally, the invention also encompasses all the equivalents to these nucleotide sequences (A) SEQ ID NOs: 2, 4, 6 and 8, which conserve the function of eIF4E genetic marker for sensitivity/resistance to potyviruses specific to the reference sequences. As regards the DNAs, they are in particular the genetic degeneracy analogs and the cDNA sequences complementary to the reference sequences. The polynucleotide equivalents of the reference sequences (A) are also found among their transcription products (RNA) (B). The proteins (C) derived from (A) and from (B) constitute other intracellular markers for selecting plants resistant or sensitive to potyviruses. Besides the targets (A), (B) and (C), the means of selection of the invention can also be nucleotide probes capable of hybridizing with complementary nucleotide targets (A) and (B), or else protein-based means of detection (antibodies D) capable of pairing with specific antigenic targets (C). It is possible to envision combining all these equivalent means (A), (B), (C) & (D) so as to form a selection tool (E).
The means according to the invention also cover any fragment of these sequences (A), (B), (C) & (D). According to the invention, the term “fragment” is intended to mean:                either a polynucleotide of at least 10, 20, 30, 50, 100, 200, 300, 400, 500 contiguous nucleotides of the reference sequence; preferred fragments are those which are capable of hybridizing selectively, under stringent conditions, with said reference sequence;        or a polyamino acid of at least 3, 6, 10, 15, 30, 60, 100, 150, 200 contiguous amino acids of the reference sequence; preferred fragments are those which are capable of hybridizing selectively, under stringent conditions, with said reference sequence.        
According to an advantageous embodiment of the invention, the method is characterized in that:                at least one means of detection comprising at least one of the tools A, B, C, D, E as claimed in claim 1 and/or at least one restriction enzyme is brought into contact with at least one genomic and/or protein extract of a plant to be tested,        said genomic and/or protein extract, optionally paired and/or hybridized and/or digested, is subjected to at least one separation,        the possible pairings and/or hybridizations and/or digestions liable to occur are revealed,        and the results are read so as to finally reach a conclusion as to the presence or the absence of an allele for resistance (pvr21) or of an allele for sensitivity (pvr+) to at least one potyvirus.        
This method falls within the framework of the methodologies known in the field of the detection and recognition of plant genetic characteristics.
According to a first embodiment of the method, in which the principle of selection is based on the use of one or more specific restriction enzymes, the method can correspond to the following methodology:                the coding sequence of the eIF4E gene is amplified by PCR from the DNA of the plant to be tested, for example using the primers SEQ ID NOs: 18 and/or 19,        the amplification product is digested with a suitable restriction enzyme,        the possible fragments obtained are separated,        and the resistant or sensitive plants are selected according to the restriction profile for said amplification product.        
For example, potyvirus-sensitive plants can be detected by means of a restriction profile which reveals the presence of a site for cleavage by the TspRI enzyme or one of its isoschizomers, and potyvirus-resistant plants can be detected by means of a restriction profile which reveals the absence of said site for cleavage with TspRI or one of its isoschizomers and the presence of a site for cleavage with the MvnI enzyme or one of its isoschizomers.
According to a second embodiment of the method, corresponding to the case where the mode of selection is the hybridization of complementary nucleotide sequences, the method preferably consists:                in extracting the DNA from plants,        in optionally subjecting this DNA to enzymatic digestion using at least one restriction enzyme,        in denaturing the optionally digested DNA,        in bringing the DNA thus denatured into contact with a probe, itself denatured beforehand and provided with at least one label, so as to carry out the hybridization,        in eliminating the DNA and the nonhybridized probe,        in revealing the hybridization by means of the label,        and in selecting plants which have a hybridization profile corresponding to cosegregation of the target hybridized with the labeled probe and of the allele for resistance or for sensitivity.        
When the DNA has been digested with a restriction enzyme, the distinction between the sensitive plants and the resistant plants can be made by means of the difference in size of the hybridized fragments.
It can also be made using a probe capable of hybridizing selectively with the allele for resistance or the allele for sensitivity. The hybridization of the single-stranded molecules of the probe and of the target is preferably carried out under stringent hybridization conditions allowing selective hybridization, which can be determined in a manner known to those skilled in the art. In general, the hybridization and washing temperature is at least 5° C. below the Tm of the reference sequence at a given pH and for a given ionic strength. Typically, the hybridization temperature is at least 30° C. for a polynucleotide of 15 to 50 nucleotides and at least 60° C. for a polynucleotide of more than 50 nucleotides.
The level of signal generated by the interaction between the sequence capable of hybridizing selectively and the reference sequences is generally 10 times, preferably 100 times, more intense than that of the interaction of the other DNA sequences generating the background noise.
With a probe labeled, for example, with a radioactive element, such as 32P, or with a grafted enzyme, such as peroxidase, the hybridization is readily revealed qualitatively and quantitatively.
The DNA used in the first or the second embodiment can be either total DNA or cDNA.
According to a third embodiment (among others) of the method according to the invention, corresponding to the case where the mode of selection is antibody/antigen pairing, the method preferably consists in detecting the presence of a polypeptide partly consisting of all or part of one of the amino acid sequences described below and included in the invention. The method may consist in bringing the sample to be tested into contact with an antibody as described above, and then in detecting the antigen/antibody complex formed.
Whatever the mode of selection, the method of selection according to the invention is reliable and sensitive.
A subject of the present invention is also a polynucleotide encoding a mutant eIF4E protein comprising a region derived from that defined by sequence (I) above, by substitution of at least one neutral amino acid of said sequence (I) with a charged amino acid, preferably a basic amino acid, and/or substitution of at least one charged amino acid of said sequence (I) with a neutral amino acid or an amino acid having an opposite charge.
According to a preferred embodiment, said polynucleotide encodes a mutant eIF4E protein which comprises a region derived from that defined by sequence (I) above, by:                a) substitution of at least one of the amino acids X1, X2, X3 or X4 of said sequence (I) with a charged amino acid, and        b) substitution of at least one of the other neutral amino acids of said sequence (I) with a charged amino acid and/or substitution of at least one charged amino acid of said sequence (I) with a neutral amino acid or an amino acid having an opposite charge.        
Polynucleotides in accordance with the invention are, for example, those which encode the variants of the sequences SEQ ID NO: 22 or 23 associated with potyvirus resistance.
According to another of its aspects, the invention concerns a nucleotide sequence characterized in that it is described by a sequence chosen from the group comprising all or some of the following sequences:                SEQ ID NO: 2        SEQ ID NO: 4        SEQ ID NO: 6        SEQ ID NO: 8.        
The nucleotide sequence SEQ ID NO: 2 is a cDNA sequence obtained from tobacco DNA and corresponding to the tobacco gene. The nucleotide sequence SEQ ID NO: 4 is a sequence encoding an eIF4E protein of a potyvirus-sensitive variety of Lycopersicon esculentum. The sequences SEQ ID NOs: 6 and 8 are sequences encoding eIF4E proteins from capsicum (Capsicum annuum), varieties Yolo Wonder and Yolo Y, respectively.
A subject of the present invention is also primers for amplifying an eIF4E gene, or a portion thereof, liable to contain at least one mutation, as defined above, associated with potyvirus resistance; they are in particular primers for amplifying the sequence of eIF4E encoding the region of eIF4E defined by sequence (I), or a mutant sequence which is derived therefrom.
Primers in accordance with the invention can be readily defined by those skilled in the art, from the nucleotide or peptide sequences described in the present invention.
By way of nonlimiting examples, mention will be made of: the amplification primers consisting of the nucleotide primer sequences SEQ ID NOs: 18 & 19; the cloning primers SEQ ID NOs: 10 to 17; the primers for screening a BAC library, consisting of the nucleotide sequences SEQ ID NOs: 20 & 21.
SEQ ID NOs: 18 & 19 are primers derived from the coding sequence of capsicum Yolo Wonder eIF4E, which make it possible, in particular by PCR amplification and then by enzyme digestion, to detect the nucleotide sequences carrying the alleles for resistance, pvr2, and for sensitivity, pvr+, to potyviruses. The degenerate cloning primers SEQ ID NOs: 10 & 11 and the nondegenerate cloning primers SEQ ID NOs: 12 to 17 were defined on the basis of an alignment of the eIF4E sequences from tobacco, tomato and Arabidopsis and used for the synthesis (RACE) of cDNA probes for detecting eIF4E in the tomato and capsicum genome. The primers SEQ ID NOs: 20 & 21 for screening a BAC library are nondegenerate. These primers SEQ ID NOs: 10 to 17, 20 & 21 can optionally be used directly or indirectly (construction of selection tools) in the detection of potyvirus resistance or potyvirus sensitivity characteristics.
A subject of the present invention is also a mutant eIF4E protein comprising a region derived from that defined by sequence (I) above, by substitution of at least one neutral amino acid of said sequence (I) with a charged amino acid, preferably a basic amino acid, and/or substitution of at least one charged amino acid of said sequence (I) with a neutral amino acid or an amino acid having an opposite charge.
According to a preferred embodiment, said mutant eIF4E protein comprises a region derived from that defined by sequence (I) above, by:                a) substitution of at least one of the amino acids X1, X2, X3 or X4 of said sequence (I) with a charged amino acid, and        b) substitution of at least one of the other neutral amino acids of said sequence (I) with a charged amino acid and/or substitution of at least one charged amino acid of said sequence (I) with a neutral amino acid or an amino acid having an opposite charge.        
The present invention also covers the products of translation of the nucleotide sequences SEQ ID NOs: 2, 4, 6 and 8, namely the polypeptides chosen from the group comprising all or some of the following sequences:                SEQ ID NO: 3        SEQ ID NO: 5        SEQ ID NO: 7        SEQ ID NO: 9.        
The means for selecting potyvirus-resistance/sensitivity of plants, consisting of amino acid sequences, are preferably used as locating targets. They are then indirect selection means which underlie the use of specific means for detecting these peptide targets.
These means of detection are advantageously antibodies which constitute another subject of the present invention. Thus, said antibodies are characterized in that they are specifically directed against all or part of one at least of the translation products C, and more particularly of the amino acid sequences SEQ ID NOs: 3, 5, 7, 9, 22 or 23 or a fragment of at least 6 amino acids thereof. These antibodies may be monoclonal or polyclonal.
The antibodies against the polypeptides as defined above can be prepared according to conventional techniques well known to those skilled in the art (for example, Kohler and Milstein, 1975; Kozbor et al. 1983, Martineau et al., 1998). An antibody according to the invention may comprise a detectable isotope or nonisotope, for example fluorescent, label or else may be coupled to a molecule such as biotin according to techniques well known to those skilled in the art.
Another section of the invention relates to means of selection made up of probes for detecting plants resistant to at least one potyvirus, these probes being taken in themselves. In this section, probes for detecting plants resistant to at least one potyvirus are defined.
A first category of probes is characterized in that each probe comprises at least one sequence corresponding to all or part of SEQ ID NOs: 2, 4, 6 and 8. Within this first category, the probes comprising at least one sequence corresponding to all or part of SEQ ID NOs: 2, 4, 6 and 8, and in particular to all or part of the portion encoding the region of the eIF4E protein defined by general sequence (I), are most especially preferred.
SEQ ID NO: 6 is a sensitivity probe derived from the capsicum Yolo Wonder. It differs from SEQ ID NO: 8, which is a resistance probe derived from the capsicum Yolo Y, by virtue of two nucleotide bases. These mutations shown on SEQ ID NOs: 6 & 8 correspond to the restriction sites TspRI for SEQ ID NO: 6 and MnvI for SEQ ID NO: 8, marking, respectively, potyvirus sensitivity in Yolo Wonder and potyvirus resistance in Yolo Y.
These probes are used to distinguish resistant and sensitive plants, either by selective hybridization and detection of the presence or of the absence of a hybridization signal, or by digestion with an appropriate restriction enzyme capable of differentially cleaving the allele for sensitivity and the allele for resistance, for example EcoRI, TspRI or MnvI, followed by hybridization of the probe with the restriction product. In the latter case, the sensitive plants are distinguished from the resistant plants by the difference in size of the hybridized fragments.
The present invention also provides tools for carrying out another method of selection in accordance with the first embodiment of the method according to the invention, as defined above. According to this first embodiment, a PCR amplification of the eIF4E sequence is first of all carried out. The amplification is followed by selective digestion with a restriction enzyme. The tools involved are therefore of two types: restriction enzyme(s) and PCR primer(s) for amplifying the eIF4E sequence.
A subject of the present invention is in particular a kit for detecting an eIF4E allele associated with potyvirus resistance or with potyvirus sensitivity, characterized in that it comprises:                at least one restriction enzyme chosen from:        a) an enzyme which recognizes a restriction site I present in at least one eIF4E allele associated with potyvirus sensitivity, and absent from the eIF4E alleles associated with potyvirus resistance;        b) an enzyme which recognizes a restriction site II present in at least one eIF4E allele associated with potyvirus resistance, and absent from the eIF4E alleles associated with potyvirus sensitivity; and        a pair of nucleotide primers for amplifying eIF4E or a portion thereof comprising the restriction site I and/or the restriction site II.        
For example,                for detecting an eIF4E allele associated with potyvirus sensitivity, such as that represented by the sequence SEQ ID NO: 6, the restriction enzyme is TspRI, or one of its isoschizomers, which recognizes a restriction site defined by the sense sequence: NNCASTGNN^ (SEQ ID NO: 24) and the antisense sequence ^NNGTSACNN (SEQ ID NO: 25). The nucleotide primers are chosen so as to allow amplification of the entire sequence of eIF4E or of at last one portion thereof comprising the TspRI site;        for detecting an eIF4E allele associated with potyvirus resistance, such as that represented by the sequence SEQ ID NO: 8, the restriction enzyme is MvnI, or one of its isoschizomers, which recognizes a restriction site defined by the sense sequence: CG^CG and the antisense sequence: GC^GC. The nucleotide primers are chosen so as to allow amplification of the entire sequence of eIF4E or of at least one portion thereof comprising the MvnI site.        
In the two cases, the primers SEQ ID NO: 18 and SEQ ID NO: 19 may, for example, be used.
As indicated above, the detection of plants sensitive or resistant to potyviruses can also be carried out by detection of the presence or of the absence of the wild-type or mutant form of the eIF4E protein.
Thus, the present invention encompasses the use of a wild-type or mutant eIF4E protein, as defined above, or of an antibody specific for one of said proteins, for selecting potyvirus-resistant plants.
Preferably, said eIF4E protein is chosen from:                the protein represented by the polypeptide sequence SEQ ID NO: 3;        the protein represented by the polypeptide sequence SEQ ID NO: 5;        the protein represented by the polypeptide sequence SEQ ID NO: 7;        the protein represented by the polypeptide sequence SEQ ID NO: 9;        the group of proteins represented by the polypeptide sequence SEQ ID NO: 22;        the group of proteins represented by the polypeptide sequence SEQ ID NO: 23.        
Thus, a category of means for detecting potyvirus resistance in accordance with the invention is characterized in that each of these means comprises at least one antibody specific for all or part of a mutant eIF4E protein in accordance with the invention, and in particular for a fragment of at least 6 amino acids thereof carrying a mutation associated with the resistance, as defined above.
For example, a means for detecting the resistance may consist of an antibody specific for all or part of a polypeptide sequence as defined above, in particular for a fragment of at least 6 amino acids thereof carrying a mutation associated with the resistance.
The invention also relates to means for detecting potyvirus sensitivity or potyvirus resistance, each consisting of at least one amino acid sequence chosen from the group comprising the following sequences:                SEQ ID NO: 3;        SEQ ID NO: 5;        SEQ ID NO: 7;        SEQ ID NO: 9;        SEQ ID NO: 22;        SEQ ID NO: 23.        
The invention relates more particularly to means for detecting potyvirus sensitivity, each consisting of at least one antibody specific for an amino acid sequence chosen from the group comprising the following sequences:                SEQ ID NO: 3;        SEQ ID NO: 5;        SEQ ID NO: 7;        SEQ ID NO: 9;        SEQ ID NO: 22;        SEQ ID NO: 23;        or a fragment of at least 6 amino acids of one of said sequences, in particular a fragment of the region thereof defined by sequence (I).        
Preferably, each nucleotide probe or any other means of detection mentioned above has at least one label, that is useful as an indicator of the nucleotide hybridization or the antigen/antibody pairing at the heart of the detection of the sensitive sequence. Advantageously, this label is detectable by spectroscopic, photochemical, biochemical, immunochemical or else chemical means. For example, such a label may consist of a radioactive isotope of 32P or 3H, of a fluorescent molecule (5-bromodeoxyuridine, fluoresceine, acetylaminofluorene) or else of a ligand such as biotin. As regards more especially the nucleotide probes, the labeling thereof is preferably carried out by incorporation of labeled molecules into the polynucleotides by primer extension or else by addition to the 3′ or 5′ ends.
Preferably, the sequences used for detecting the potyvirus-resistant plants are used as nucleotide probes or primers.
It goes without saying that not all the abovementioned means of detection are limited strictly to the denoted sequences, but encompass all the equivalents consisting in particular of the similar sequences which conserve the function under consideration, and as defined above.
Those skilled in the art are fully aware of the various methods for preparing probes and primers, including by cloning and by the action of restriction enzymes, or else by direct chemical synthesis according to techniques such as the phosphodiester method of Brown et al. (1979) or the solid-support technique described in European patent No. EP 0707592. The nucleic acids can be labeled, if desired, by incorporating a detectable molecule or label as set out above. Examples of nonradioactive labeling of nucleic acid fragments are described in particular in French patent No. FR 78 10 975 or else in the articles by Urdéa et al., (1988) or Sanchez-Pescador et al. (1988).
According to another of its aspects, the present invention relates to the use of the means of detection defined above, for detecting plants resistant/sensitive to at least one potyvirus.
In accordance with the invention, the MvnI and/or TspRI restriction sites, which are incidentally known, of the eIF4E sequences are used as oligonucleotide marker(s) for potyvirus resistance/sensitivity.
Preferably, the restriction sites used as oligonucleotide marker(s) correspond:                to the sense sequence: CG^CG and to the antisense sequence: GC^GC        and/or the sense sequence: NNCASTGNN^ (SEQ ID NO: 24) and to the antisense sequence ^NNGTSACNN (SEQ ID NO: 25).        
The use of these restriction sites as markers (or labels or tags) for potyvirus resistance on expressed sequences ties up with the first embodiment of the method of detection described above, in which use is made of restriction enzymes (for example: MvnI and/or TspRI) and of primers for amplification of the eIF4E sequence, for example: SEQ ID NO: 18 and/or 19.
Considering the specificity of the MvnI & TspRI restriction sites, the present invention also encompasses the sue, as oligonucleotide marker(s) for potyvirus resistance/sensitivity, of the abovementioned MvnI & TspRI restriction sites and, preferably, of the restriction site corresponding to the sense sequence: CG^CG and to the antisense sequence GC^GC and/or of the restriction site corresponding to the sense sequence: NNCASTGNN^ (SEQ ID NO: 24) and to the antisense sequence ^NNGTSACNN (SEQ ID NO: 25).
According to another of its aspects, the present invention relates to a kit for selecting potyvirus-resistant/sensitive plants, comprising at least one means of detection of antibody or polynucleotide type as defined above. The kit comprises, where appropriate, the reagents required for carrying out a hybridization or amplification reaction.
A subject of the invention is also the plants derived from the method described above and/or from the implementation of the tools and/or of the use and/or of the selection kit defined above. Preferably, these plants belong to the family of the Solanaceae, Cucurbitaceae, Cruciferae and Compositae. Even more preferably, they are chosen from tomatoes, capsicums and/or lettuce.
By way of examples, the inventors carry out the method which is the subject of the invention by following the protocol for RFLP analysis (restriction fragment length polymorphism). To do this, the inventors have used a conventional RFLP protocol in which the probes which are the subject of the invention are labeled with 32P and in which the DNA from the capsicum plants to be analyzed is digested with the EcoRI restriction enzyme. At the end of this method, the inventors obtain hybridization profiles which are different between the potyvirus-resistant plants and the potyvirus-sensitive plants, thus making it possible to select the sensitive or resistant plants. The latter may then enter into a program of plant improvement by successive crossing.
The invention does not concern only the selection of plants resistant or sensitive to potyviruses. In fact, insofar as the inventors have been able to identify the eIF4E gene determining a recessive resistance to potyviruses, it is from now on possible to envision obtaining new varieties of genetically modified plants that are resistant (or sensitive) to at least one potyvirus.
The invention therefore relates to a nonbiological method for obtaining new varieties of genetically modified plants that are resistant (or sensitive) to at least one potyvirus, characterized in that it consists essentially in seeing to it that an eIF4E allele associated with resistance (or with sensitivity) to said potyvirus appears in the genome of these plants, and/or in introducing said allele into the genome of these plants.
According to an advantageous embodiment of this method, the appearance of the resistance allele is brought about by implementing a method selected from the group comprising:                mutagenesis, advantageously “tilling”,        homologous recombination,        overexpression,        insertion/deletion,        gene silencing/transgenesis,        and combinations thereof.        
The tools which may be used in the abovementioned nonbiological method of production also form an integral part of the present invention.
A subject of the present invention is thus any constructed genetic unit comprising a polynucleotide in accordance with the invention encoding a mutant eIF4E protein, placed under the control of suitable elements for controlling the transcription and, optionally, the translation.
Said mutant eIF4E protein can advantageously be chosen from the variants of the sequences SEQ ID NO: 22 or 23 associated with potyvirus resistance.
A subject of the present invention is also any constructed genetic unit, characterized in that it comprises:                at least one genetic tool A/ and/or B/ as defined above,        and/or at least one nucleotide sequence chosen from the group comprising all or part of the following sequences:                    SEQ ID NO: 6            SEQ ID NO: 8,                        and/or at least one nucleotide sequence encoding an eIF4E factor and comprising at least one MvnI and/or TspRI restriction site, and preferably at least one restriction site corresponding to the sense sequence: CG^CG and to the antisense sequence: GC^GC and/or a restriction site corresponding to the sense sequence: NNCASTGNN^ and to the antisense sequence: ^NNGTSACNN.        
Another tool for genetic transformation covered by the invention consists of any vector for transforming plant cells, comprising at least one constructed genetic unit as mentioned above. It may be any known and appropriate cloning vector (phages, plasmids, cosmids, etc.).
The plant cells and the microorganisms transformed by means of at least one vector or of at least one constructed genetic unit as defined above are also part of the invention.
At a higher level, the invention encompasses the plants transformed by means of at least one vector and/or of at least one constructed genetic unit and/or of transformed plant cells and/or of transformed microorganisms, as they have been described above.
Those skilled in the art are very aware of all the direct or indirect techniques for genetic modification. Additional details are given in the examples which follow.